The goal of this proposal is to define the mechanisms involved in the generation of human adenovirus-specific cytotoxic T lymphocytes (CTLs) in order to help design new strategies for evading the immune response to adenovirus gene therapy vectors. Adenoviruses are under extensive investigation as gene therapy vectors for a broad spectrum of heart, lung, and blood diseases including cystic fibrosis, hemophilia, and atherosclerosis. However, data from animal models indicate that the immunogenicity of adenovirus vectors interferes with the efficacy of adenovirus-mediated gene therapy. Administration of early region 1(E1)-deleted adenovirus vectors to mice, a host in which adenovirus infection is naturally restricted, results in the generation of adenovirus-specific CTLs which destroy adenovirus-transduced cells within a few weeks. Further analysis of this problem requires study of human CTL responses against adenovirus. We have successfully amplified memory adenovirus-specific CTLs in peripheral blood mononuclear cells from healthy adults and documented that these responses are major histocompatiblity complex (MHC)-restricted and mediated by CD8+ T cells. Based on our studies, it is likely that the presence of memory cellular immune responses will pose a major additional obstacle for adenovirus-mediated gene therapy in man. We postulate, however, that human adenovirus-specific CTLs may be targeted against a limited number of immunodominant epitopes. Therefore, it may be possible to reduce the immunogenicity of adenovirus vectors by elimination of such epitopes. As a second approach, it may be possible to take advantage of mechanisms which adenovirus has developed to evade host immune responses. The Ad early region 3 (E3) codes for proteins which help make cells resistant to CTLs and tumor necrosis factor, but this region is deleted or poorly expressed from most adenovirus vectors. We postulate that constitutive expression of E3 region proteins may help reduce the immunogenicity of adenovirus vectors. We propose to address these hypotheses by analysis of human CTL responses against adenovirus in vitro. These studies will provide an experimental basis for the design of more effective adenovirus gene therapy vectors for future human trials.